Metabolic engineering of Corynebacterium glutamicum for production of the adipate precursor 2-oxoadipate

摘要

The C6-dicarboxylic acid adipate is one of the most important building blocks in the chemical industry. The chemical syntheses of adipate are connected to insanitary and environmentally harmful reagents and catalysts. Therefore, the establishment of a biotechnological process for adipate production based on renewable carbon sources is of substantial interest. Thus, the main objective of this work was the metabolic engineering ofCorynebacterium glutamicum for the production of the adipate precursor 2-oxoadipate. This organism is an industrially established producer of amino acids, but was also shown to possess a high capability for the production of organic acids, such as lactate or succinate. Neither adipate nor 2-oxoadipate (solutions adjusted to pH 7) were metabolised by C. glutamicum wild type and they did not inhibit growth in glucose minimal medium in concentrations up to 50 mM (μ = 0.43 h-1). Addition of 150 mM or 250 mM adipate resulted in a 37% decreased growth rate (μ = 0.29 h-1), whereas a concentration of 500 mM adipate almost completely abolished growth. When adjusting the pH of the adipate solution with KOH to pH 7, the addition of 500 mM adipate still allowed a growth rate μ of 0.09 h-1. Additional supplementation of 5 mM of the compatible solute L-proline further increased the growth rate to 0.16 h-1. The latter result and DNA microarray experiments performed in the presence of 50 mM and 150 mM adipate indicated that the growth retardation might be causedto some extent by osmotic stress. Furthermore, the transcriptome data revealed high upregulation of the transcriptional regulator PcaR and its target genes pcaIJ and pcaFDO, belonging to the PCA (protocatechuate) branch of the β-ketoadipate pathway. Thus, adipate probably acts as an activator molecule of PcaR in C. glutamicum. ...